Rich spatiotemporal dynamics of cortical activity, including complex and diverse wave patterns, have been identified during unconscious and conscious brain states. Yet, how these activity patterns emerge across different levels of wakefulness remain unclear. Here we study the evolution of wave patterns utilizing data from high spatiotemporal resolution optical voltage imaging of mice transitioning from barbiturate-induced anesthesia to wakefulness (N = 5) and awake mice (N = 4). We find that, as the brain transitions into wakefulness, there is a reduction in hemisphere-scale voltage waves, and an increase in local wave events and complexity. A neural mass model recapitulates the essential cellular-level features and shows how the dynamical competition between global and local spatiotemporal patterns and long-range connections can explain the experimental observations. These mechanisms possibly endow the awake cortex with enhanced integrative processing capabilities.

Sensory inputs carry critical information for the survival of an organism. In mice, tactile information conveyed by the whiskers is of high behavioural relevance, and is broadcasted across cortical areas beyond the primary somatosensory cortex. Mesoscopic voltage sensitive dye imaging (VSDI) of cortical population response to whisker stimulations has shown that seemingly ‘simple’ sensory stimuli can have extended impact on cortical circuit dynamics. Here we took advantage of genetically encoded voltage indicators (GEVIs) that allow for cell type-specific monitoring of population voltage dynamics in a chronic dual-hemisphere transcranial windowed mouse preparation to directly compare the cortex-wide broadcasting of sensory information in wakening (lightly anesthetized to sedated) and awake mice. Somatosensory-evoked cortex-wide dynamics is altered across brain states, with anatomically sequential hyperpolarising activity observed in the awake cortex. GEVI imaging revealed cortical activity maps with increased specificity, high spatial coverage, and at the timescale of cortical information processing.

Rapidly progressing development of optogenetic tools, particularly genetically encoded optical indicators, enables monitoring activities of neuronal circuits of identified cell populations in longitudinal in vivo studies. Recently developed advanced transgenic approaches achieve high levels of indicator expression. However, targeting non-sparse cell populations leads to dense expression patterns such that optical signals from neuronal processes cannot be allocated to individual neurons. This issue is particularly pertinent for the use of genetically encoded voltage indicators whose membrane-delimited signals arise largely from the neuropil where dendritic and axonal membranes of many cells intermingle. Here we address this need for sparse but strong expression of genetically encoded optical indicators using a titratable recombination-activated transgene transcription to achieve a Golgi staining-type indicator expression pattern in vivo. Using different transgenic strategies, we also illustrate that co-expression of genetically encoded voltage and calcium indicators can be achieved in vivo for studying neuronal circuit input–output relationships.

Cellular resolution voltage imaging enables direct observation of neuronal computation. Indeed, membrane potential imaging experiments have spatiotemporally resolved both active and passive action, and synaptic potential generation throughout dendritic and axonal arbors.^{1 – 14} Resolution of these small voltage signals at high speeds requires high photon fluxes, making wide-field single-photon (1P) imaging by far the most common voltage imaging modality. Imaging neuronal processes with this technique require the imaged membranes to lie approximately flat in the microscope’s focal plane. As these experiments are typically performed in slices, the requirement for flat, healthy, and superficial cells represents a significant barrier to entry for experimenters. Even in the best-prepared slices, anatomy dictates that only a few cells will be oriented parallel to the surface, reducing experimental throughput, and only certain cell types feature morphology that can be well-sampled by a single plane. Iterative three-dimensional (3-D) imaging in wide field via physical refocusing is often untenable due to rapid sample bleaching. Multiple approaches to improve wide-field imaging’s 3-D performance have been developed. Anselmi et al.¹⁵ applied remote focusing to axially shift and tilt the wide-field focal plane as required by the sample, enabling calcium imaging along tilted dendrites. This adaptation, however, costs half of the fluorescence emission and is limited to a single tilted plane at a time. Point spread function (PSF) engineering via cubic phase masks¹⁶ or spherical aberration¹⁷ also enables parallelized volumetric sample imaging when combined with light-sheet excitation; however, to our knowledge, these approaches have not successfully been implemented to image membrane voltage.

DNA methylation profiling has become a promising approach towards identifying biomarkers of neuropsychiatric disorders including autism spectrum disorder (ASD). Epigenetic markers capture genetic risk factors and diverse exogenous and endogenous factors, including environmental risk factors and complex disease pathologies. We analysed the differential methylation profile of a regulatory region of the GAD1 gene using cerebral organoids generated from induced pluripotent stem cells (iPSCs) from adults with a diagnosis of ASD and from age- and gender-matched healthy individuals. Both groups showed high levels of methylation across the majority of CpG sites within the profiled GAD1 region of interest. The ASD group exhibited a higher number of unique DNA methylation patterns compared to controls and an increased CpG-wise variance. We detected six differentially methylated CpG sites in ASD, three of which reside within a methylation-dependent transcription factor binding site. In ASD, GAD1 is subject to differential methylation patterns that may not only influence its expression, but may also indicate variable epigenetic regulation among cells.